Chromatography may generally be used to separate compounds from a mixture. For example, in liquid chromatography, chromatography relies on compounds having a different distribution between solid particles in a stationary phase of a column and a liquid phase that is passed through the column. For ideal utilization of chromatography, the column packing material needs to be uniformly packed to provide a consistent path length for the separations to occur. A specific compound's affinity for the column packing material compared to its affinity for the mobile phase passing through the column determines the amount of time that the compound resides inside the column. After the compounds exit the column, they may be either individually detected (e.g. analytical liquid chromatography) or individually collected (e.g. preparative liquid chromatography). For the chromatographic separations to be carried out efficiently, the column packing material must be uniformly dispersed in the column with no gaps or cracks that would create re-mixing of the separated compounds.
The use of particles smaller than 2 μm in diameter has improved the performance of chromatographic separations (J. Chromatog. 1127: 60-69, 2006). A significant limitation to the chromatographic performance of sub-2 μm particles is the difficulty in achieving uniform packing; packed columns exhibit radial heterogeneity in packing, which deteriorates performance. This effect is called eddy diffusion. For example, a 100 μm inner diameter (i.d.) column of packed silica particles exhibits a contribution of 1.0 μm to the length-normalized peak variance (commonly called height equivalent to a theoretical plate) due to eddy diffusion (Anal. Chem. 76: 5777-5786, 2004). If the silica particles could be packed uniformly, i.e., with radial homogeneity, then separation performance would be expected to continue to improve as the particles decreased in size. Such improvements would be valuable, especially for protein and peptide separations, where there is a demand for separating mixtures of thousands of components, resolving very similar isoforms of a single protein, or determining the purity of therapeutic monoclonal antibodies. This improvement in packing would enable the commercial use of submicrometer particles for these and other separations. Silica particles have been successfully packed with virtually crystalline order in capillaries by using submicrometer, nonporous silica particles that have a narrow size distribution (Langmuir 20: 2033-2035, 2004). The limitation with this method is that there are usually gaps where the material meets the capillary wall, obviating any improvements in performance, and the packing procedure takes days.